Physical adaptation is a hallmark of human evolution. Morphological changes in our limbs allowed us to make and use sophisticated tools and to walk upright, and the expansion of the human cortex is the origin of our advanced cognitive abilities. These traits are ultimately encoded in genetic changes that arose during human evolution, and which acted to alter molecular and cellular processes during development. The goal of this ongoing research project, which began in 2010, is to determine where in the genome those changes reside and to understand their biological functions. Our efforts focus on two classes of gene regulatory elements that may encode novel functions in humans. The first are Human Accelerated Regions (HARs), many of which encode transcriptional enhancers that are highly conserved across species but show multiple human-specific sequence changes. The second class of elements are Human Gain Enhancers (HGEs), which are transcriptional enhancers that show increased activity in developing human tissues based on comparisons of epigenetic marks associated with enhancer activity in human, rhesus macaque and mouse. These discoveries reveal that changes in developmental gene regulation played a central role in the evolution of uniquely human morphology and provide the means to experimentally model the evolution of human development. In this funding cycle, we will use humanized mouse models to study the biological function of HACNS1, which we identified as the first known HAR to encode human-specific regulatory activity. HACNS1 maintains its human-specific activity in the developing mouse limb and alters the expression of the nearby transcription factor Gbx2 in the developing embryo. We hypothesize that HACNS1 is acting within a larger network of human-specific regulatory changes that modified development. Using the HACNS1 mouse model and our maps of human-specific regulatory functions in the limb as entry points, we will identify transcriptional and regulatory changes downstream of HACNS1 at single-cell resolution, model additional human-specific regulatory functions that may interact with the regulatory changes driven by HACNS1, and determine how these changes influence the development of the limb.